Phospholipid scrambling by a TMEM16 homolog of Arabidopsis thaliana

Membrane asymmetry is important for cellular physiology and established by energy‐dependent unidirectional lipid translocases, which have diverse physiological functions in plants. By contrast, the role of phospholipid scrambling (PLS), the passive bidirectional lipid transfer leading to the break‐down of membrane asymmetry, is currently still unexplored. The Arabidopsis thaliana genome contains a single gene (At1g73020) with homology to the eukaryotic TMEM16 family of Ca2+‐activated phospholipid scramblases. Here, we investigated the protein function of this Arabidopsis homolog. Fluorescent AtTMEM16 fusions localized to the ER both in transiently expressing Arabidopsis protoplasts and HEK293 cells. A putative scrambling domain (SCRD) was identified on the basis of sequence conservation and conferred PLS to transfected HEK293 cells, when grafted into the backbone of the non‐scrambling plasma membrane‐localized TMEM16A chloride channel. Finally, AtTMEM16 ‘gain‐of‐function’ variants gave rise to cellular phenotypes typical of aberrant scramblase activity, which were reversed by the additional introduction of a ‘loss‐of‐function’ mutation into the SCRD. In conclusion, our data suggest AtTMEM16 works as an ER‐resident lipid scramblase in Arabidopsis.


Introduction
Providing a physical boundary towards the external environment and between the different compartments of eukaryotic cells, biological membranes are important interfaces for the exchange of matter, energy and information. As such, their lipid and protein composition is tailored to their specific physiological functions and determines their identity. Membrane lipids show significant chemical and organizational complexity. Individual lipid species of a membrane vary in their amount, in their localization in subdomains within a membrane leaflet, for example, lipid rafts or nanodomains, and finally also in their relative distribution between the two leaflets of the bilayer. This membrane asymmetry is an important prerequisite for many cellular processes, for example, for the generation of membrane curvature during vesicle-mediated membrane trafficking, membrane-based signalling events and the recruitment of proteins to specific membrane domains or organelles. In plants, anionic phospholipids like phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) are highly enriched at the cytosolic face of the plasma membrane and, to a lower extent, down the endocytic pathway [1,2]. It has been proposed that the graded distribution of negative charge provided by their anionic head groups determines an electrostatic territory attracting positively charged interactors from the cytosol [3][4][5].
The establishment and maintenance of phospholipid asymmetry is an energy-consuming process. The spontaneous inter-leaflet movement of phospholipids is very slow [6], since the translocation of the polar head groups across the hydrophobic membrane core is energetically unfavourable. Lipid flippases are primary active transporters moving phospholipids against a concentration gradient to the cytosolic side of the membrane. Most flippases belong to the P4 ATPase subfamily of P-type ATPases and plant genomes generally contain a large number of P4 ATPase homologs [7], and also in the model plant Arabidopsis thaliana 12 family members have been identified [8], which were named aminophospholipid ATPases (ALA). Based on the analysis of gene knock-out mutant plants, ALA proteins have been implicated in developmental processes, cellular signalling, defence responses, and adaptation to temperature changes (recently reviewed by Nintemann et al., [9]). As noted by these authors, such pleiotropy in flippase mutant phenotypes might be indicative of a non-specific impairment of cellular lipid homeostasis and/ or vesicle trafficking. The widespread expression pattern of ALA proteins and their localization in the plasma membrane as well as different endomembrane systems [9], consistent with an asymmetric character for these membranes, support the view that membrane asymmetry may be the default state in plant cells.
Just as the asymmetric distribution of phospholipids has functional relevance for specific cellular processes, so has also the rapid regulated break-down of lipid asymmetry (lipid scrambling). For comparison, the transmembrane proteins mediating this passive equilibrative lipid transport (lipid scramblases) in this context correspond to passive ion channels harvesting the energy contained in the electrochemical gradient created by primary active ion pumps (which in turn are analogous here to lipid flippases). In animal cells, the extracellular exposure of PS, normally confined to the internal leaflet of the plasma membrane, is a critical signal involved in blood coagulation or the recognition of apoptotic cells [10]. At present, the available evidence for lipid scrambling in plants is very limited. In suspension-cultured tobacco cells, induction of apoptosis evoked the binding of the PS-marker annexin-V at the cell surface [11], which suggests extracellular PS exposure may occur also in plants. Furthermore, constitutive and passive phospholipid translocation activity was identified in spinach ER membrane extracts [12], which may serve to maintain membrane symmetry and stability, since the de novo synthesis of phospholipids occurs at the cytoplasmic leaflet. No specific gene product has been assigned to these activities.
TMEM16 proteins (also named anoctamins) are a family of integral membrane proteins present in all eukaryotes. Several members of animal and fungal origin have been shown to function as Ca 2+ -dependent lipid scramblases [13][14][15][16][17][18]. Most eukaryotic genomes contain only one or two TMEM16 homologs, while the mammalian subfamily has diversified into 10 members, some of which localize to the plasma membrane [17,19] and others intracellularly [14,15,20]. Intriguingly, two of them, TMEM16A and TMEM16B, have lost their ability to scramble lipids and evolved into Ca 2+ -dependent chloride channels, which are critically involved in different aspects of epithelial transport, smooth muscle function and neuronal signalling [21]. Since the chloride-channel function has not been observed outside the mammalian subfamily, it may be legitimate to consider lipid scrambling the original function of TMEM16 proteins. During evolution, this transition from lipid channel to ion channel may have been facilitated by the fact that all recognized scramblases give rise to Ca 2+ -activated non-selective ion currents [15,16,[22][23][24], most likely due to unspecific ion slippage along the lipid transport pathway. Structural analyses have shown that TMEM16 scramblases are homodimers with a double-barrelled architecture, each monomer having 10 transmembrane-spanning helical domains and an open groove exposed to the hydrophobic membrane core as a lipid conduction pathway [13,14,25].
The genome of the model plant Arabidopsis thaliana contains a single TMEM16-like gene (locus At1g73020). Among other putative anion transporter candidates, AtTMEM16 has been included in a recent study investigating anion transport across the plasma membrane of Arabidopsis pollen tubes. Pollen tube protoplasts from tmem16 knock-out plants showed specific alterations of their membrane currents, which lead the authors to propose the AtTMEM16 protein as an anion/H + cotransporter [26]. However, direct data on protein function in heterologous expression systems and on its subcellular localization are still missing. Here, we show that At-TMEM16 localizes to the ER membrane and likely functions as a Ca 2+ -dependent lipid scramblase.

Results and discussion
The AtTMEM16 protein sequence presents features of Ca 2+ -activated lipid scramblases To obtain cues on the putative function of At-TMEM16, we first examined its predicted amino acid sequence. The AtTMEM16 gene (At1g73020) contains an open reading frame encoding 665 amino acids. Overall sequence identity with TMEM16 proteins from other kingdoms is low (Fig. S1) 25.5% with human TMEM16K (660 aa). Important identifying features of mammalian TMEM16 proteins are their cytosolic calcium dependence and their functional dichotomy into chloride channel and phospholipid scramblase branches. The ability to scramble in the latter has been nailed down to a short protein stretch spanning the cytosolic face membrane between TM4 and TM5, denominated scrambling domain (SCRD) [18]. Notably, using SCRD swapping it has been possible to convert the non-scrambling TMEM16A chloride channel into a scrambler [18,27].
Sequence alignments of the SCRD homology region for several mammalian and fungal TMEM16 proteins of demonstrated function (Fig. 1A) showed that AtTMEM16 shares two amino acid residues, glutamate at SCRD position 15 and lysine at position 30, which are fully conserved in both scramblases and chloride channels. Gyobu et al. (2017) identified two key residues contributing to the scrambling activity of TMEM16E, also present in TMEM16F, that is, lysine at SCRD position 6 and serine at position 26. Both residues are well conserved among scramblases of the murine and human TMEM16 families, and TMEM16E mutants carrying the corresponding residues present in the chloride channel TMEM16A lost A B Fig. 1. Scrambling domain sequence alignment of TMEM16 family members. Protein sequence alignments of the 35-aa scrambling domain (SCRD) and adjacent regions (in grey letters) of AtTMEM16 with TMEM16 homologs of known function (A) and with TMEM16 homologs from different dicot and monocot plant species (B). In (A), sequences of the human lipid scramblases TMEM16E, TMEM16F and TMEM16K, the fungal lipid scramblases afTMEM16 and nhTMEM16, and the human Clchannels TMEM16A and TMEM16B. In (B), sequences from Brassica rapa (Brassicaceae), Glycine max (soybean; Fabaceae), Solanum lycopersicum (tomato; Solanaceae), Vitis vinifera (grape; Vitaceae), Malus domestica (apple; Rosaceae), Papaver somniferum (Papaveraceae), Musa acuminata (banana; Musaceae), Oryza sativa (rice), Zea mays (maize) and Brachypodium distachyon (all three from Poaceae). Amino acid residues fully conserved among TMEM16 homologs are marked in grey. Two critical amino acid residues for HsTMEM16E scramblase function [28] are marked in yellow (lysine at position 6) and red (serine at position 26). Inner gate residues homologous to Phe518 in murine TMEM16F [36] are marked in cyan. Fully conserved SCRD amino acid residues within the plant TMEM16 group are indicated by an asterisk. their scrambling activity [28]. Moreover, isoleucine substitution of the serine residue in human TMEM16E (Ser555Ile), related to limb-girdle muscular dystrophy [29], a recessively inherited genetic disease, abolished both non-selective ion transport and lipid scrambling [30]. Notably, both scramblingrelated residues are also present at the homologous positions of AtTMEM16 (Fig. 1A) and fully conserved in the SCRD sequences from ten further dicot and monocot plant species (Fig. 1B), suggesting that the respective amino acid positions were not subject to variation during recent plant evolution. All TMEM16 proteins investigated so far require increased cytosolic Ca 2+ concentrations for activation. The primary Ca 2+ -binding pocket present in both fungal and animal homologs is formed by three pairs of amino acid residues located at the inner parts of TM domains 6, 7, and 8 [13,14,25,31]. Sequence alignments of these regions showed that the four conserved acidic residues at positions 2-5 (either glutamate or aspartate) were present also in AtTMEM16 ( Fig. 2A). Conversely, AtTMEM16 carries a tryptophan residue at position 1, which generally displays some intra-and inter-specific variability, and an asparagine residue instead of the highly conserved aspartate at position 6 ( Fig. 2A). Interestingly, both these sequence variations in AtTMEM16 were confirmed in further species of the Brassicaceae family (with Brassica rapa shown in Fig. 2 being one example), but not in more distant plant relatives, which without exception carry a glutamate residue at position 1 and an aspartate residue at position 6 ( Fig. 2B). In addition to this primary binding pocket for 2 Ca 2+ ions, very recently a 'third Ca 2+ site' has been identified in mammalian TMEM16A and TMEM16K [14,32]. Also, this site appears to be well conserved in AtTMEM16 (Fig. 2C), albeit with a plant-specific glutamate to glutamine exchange at position 1 ( Fig. 2C-D). In summary, the protein sequence of AtTMEM16 (and of plant TMEM16 homologs in general) shows key features of Ca 2+ -activated lipid scramblases.

AtTMEM16 shows ER localization in Arabidopsis mesophyll cells and HEK293 cells
Several members of the mammalian TMEM16 protein family are targeted to the plasma membrane (PM) [17,19,33], while others retain an intracellular localization [14,15,20]. To determine the subcellular localization of the plant homolog, fluorescently tagged AtTMEM16 proteins were transiently expressed in isolated Arabidopsis mesophyll cells. Confocal imaging showed that AtTMEM16-EGFP expression gave rise to patchy fluorescence signals, which appeared mostly intracellular and were particularly intense in the regions surrounding the chloroplasts (Fig. 3A). The space occupied by the large lytic vacuole remained unlabelled in these protoplasts. Coexpression of the mCherry-HDEL marker showed that this EGFPpositive compartment corresponded largely to the endoplasmic reticulum (Fig. 3B). By contrast, no fluorescence overlap was observed in protoplasts stained with the PM marker FM4-64 (Fig. 3C). Similar results were obtained with an AtTMEM16 fusion protein carrying yellow fluorescent protein (YFP) at its Nterminus ( Fig. 3D-F). For quantitative analyses of AtTMEM16 and ER marker co-localization, we determined Manders' overlap coefficient representing the fraction of AtTMEM16-containing pixels which were additionally positive for ER marker: M values were 0.69 AE 0.03 for AtTMEM16-EGFP (n = 19) and 0.78 AE 0.04 for YFP-AtTMEM16 (n = 22). A number of studies have shown that human embryonic kidney (HEK293) cells represent a valid heterologous expression system for functional studies on TMEM16 ion channels and lipid scramblases [15,24,30,[34][35][36][37][38]. In transiently transfected HEK293  cells, EGFP-tagged AtTMEM16 localized to intracellular membranes, showing near-complete colocalization with the ER marker CellLight ER-RFP ( Fig. 4A; M = 0.82 AE 0.03, n = 11). By contrast, no overlap with the FM4-64 marker was apparent suggesting that AtTMEM16 is not trafficked to the PM in these cells (Fig. 4B). To test the possibility that a minor fraction of AtTMEM16 protein may still be present at the PM, we performed whole-cell patchclamp recordings with an intracellular solution containing 3 µM free Ca 2+ , sufficient to activate nonselective ion currents mediated by human TMEM16E (Fig. 4C), an intracellular lipid scramblase with partial PM localization in HEK293 cells [15]. However, in AtTMEM16-EGFP expressing cells, current amplitudes were low and not significantly different from those seen in non-transfected control cells (Fig. 4C), indicating that AtTMEM16 is not trafficked to the PM, as anticipated from confocal data, or alternatively, is unable to mediate ion currents under these conditions. Taken together, these data indicate that AtTMEM16 retains an intracellular localization in the ER compartment of both Arabidopsis mesophyll protoplasts and cultured HEK293 cells.
The AtTMEM16 SCRD confers scrambling activity to the TMEM16A chloride channel Lack of PM localization precluded a direct investigation of the putative lipid scrambling function of At-TMEM16. We, therefore, applied an established chimeric approach [18,27] consisting in swapping the 35-aa SCRD homology region of AtTMEM16 (amino acids Thr366 to Tyr400; Fig. 1) into the backbone of the PMlocalized Ca 2+ -activated chloride channel TMEM16A (denominated TMEM16A-AtSCRD). We used HEK293 live-cell assays of PM-localized lipid scrambling activity, based on the binding of Alexa555-conjugated annexin-V to extracellularly exposed phosphatidylserine [15,30]. To stimulate cytosolic Ca 2+ increases required for TMEM16 protein activation, cells were acutely exposed to the Ca 2+ ionophore A23187. No Alexa555-related fluorescence signals were detected at the surface of AtTMEM16-EGFP expressing cells in any condition ( Fig. 5A), as expected from the above results showing AtTMEM16 absent from the PM (Fig. 4). Murine TMEM16A-EGFP fusion protein localized to the PM, as reported earlier [19], but was also unable to support Ca 2+ -activated lipid scrambling (Fig. 5B).
We observed that EGFP signals in TMEM16A-AtSCRD expressing cells were prevalently intracellular (Fig. 5C), indicating that the PM localization of the chimera was impaired to some extent compared to the TMEM16A wild-type protein. Notably, however, these cells presented clear Alexa555 fluorescence signals along their cell boundaries, when extracellular Ca 2+ entry was stimulated by ionophore treatment (Fig. 5C). As mentioned above, scrambling activity of mammalian TMEM16E critically depends on serine residue 555 located inside the 35-aa SCRD [28,30]. In particular, the Ser555Ile substitution related to limbgirdle muscular dystrophy [29] was recently shown to cause loss of TMEM16E function [30]. Protein sequence alignments showed that a serine residue is present at the homologous position of AtTMEM16, Ser391 (Fig. 1A). Notably, when the Ser391Ile substitution was introduced into the TMEM16A-AtSCRD chimera, annexin-V binding to transfected HEK293 cells was abolished (Fig. 5D). These results suggest that the AtTMEM16 SCRD was sufficient to confer lipid scrambling activity on the TMEM16A chloride channel, which relied on the presence of the conserved Ser391 residue.

Rescue of a gain-of function phenotype by a single mutation in the AtTMEM16 SCRD
Finally, we used a novel type of functional read-out for lipid scrambling activity of AtTMEM16, based on the incidence of a round-shaped phenotype among transfected HEK293 cells [30]. Several earlier studies have shown that cultured HEK293 or COS-7 cells transiently expressing TMEM16 mutants with constitutive scrambling activity changed their morphology from elongated-fusiform to round-shaped and lost their surface adhesion [15,30,36,39]. All tested TMEM16E gainof-function mutations causing the human bone disorder gnathodiaphyseal dysplasia (GDD) correlated with high percentages of round-shaped HEK293 cells, a phenotype which could be rescued by the additional introduction of a loss-of-function mutation [30]. Regrettably, none of the amino acid residues affected in TMEM16E GDD mutants is conserved in the AtTMEM16 protein sequence. Therefore, in order to create a hyperactive AtTMEM16 protein, we first made use of the recent identification of an inner activation gate in TMEM16 scramblases and ion channels, constituted by hydrophobic amino acid residues in the middle of the lipid permeation pathway [36]. Interestingly, changing Phe518 in murine TMEM16F (or Leu543 in murine TMEM16A) into a positively charged lysine residue promoted constitutive lipid scrambling activity independent of cytosolic Ca 2+ binding [36]. AtTMEM16 carries a leucine residue (Leu359) at the homologous position (Fig. 1A). Upon transfection with an AtTMEM16-EGFP construct carrying the Leu359Lys mutation, we observed that EGFP-positive HEK293 cells gradually changed from morphologically normal in an early stage (Fig. 6A, top) to a round-shaped and detached phenotype in later stages (Fig. 6A, bottom). In the time window 24-30 h after transfection, the percentage of round-shaped cells among all transfected cells was 46 AE 2% on average (Fig. 6B), compared to 14 AE 1% for wild-type AtTMEM16, which was very close to the value determined for wild-type TMEM16E [30]. We observed a similar round-shaped cell percentage for a second mutant, Leu640Ala (44 AE 3%; Fig. 6B). The Leu640 residue is located in the middle of a 9-aa stretch in the AtTMEM16 C-terminus, which is highly conserved among plant TMEM16 proteins (Fig. 6C). Elevated round-cell percentages-indicative of aberrant scrambling activity -for the Leu640Ala mutant suggested that the C-terminal region may have an as yet unidentified regulatory role in TMEM16 protein activity. This is also supported by the fact that serine substitution of Phe930 in the C-terminus of human TMEM16A gave rise to constitutively active chloride channels [40]. Notably, in both Leu359Lys and Leu640Ala mutants, the additional introduction of the Ser391Ile 'loss-of-function' mutation into the respective mutant background effectively suppressed the round cell phenotype (L359K/S391I: 24 AE 2%; L640A/S391I: 18 AE 1%; Fig. 6B), indicating that modified At-TMEM16 was responsible for aberrant lipid scrambling in intracellular compartments and required the conserved Ser391 residue in its SCRD for this activity.

Conclusions
The data presented here suggest that AtTMEM16, a plant homolog of eukaryotic TMEM16 proteins, possesses lipid scrambling activity, taking place in intracellular compartments, presumably in the endoplasmic reticulum. Mammalian TMEM16 scramblases with similar localization are TMEM16E [15,20], TMEM16H [41], and TMEM16K [14], yet our picture of their specific roles in cellular physiology is still blurred. One possibility is that they contribute to the maintenance of membrane symmetry in the ER, where phospholipids are newly synthesized at the cytoplasmic leaflet. In keeping with this idea, the presence of TMEM16K was required for Ca 2+ -induced phosphatidylserine redistribution within the ER membrane of mouse fibroblasts [42]. Intriguingly, recent studies further suggested tethering roles for TMEM16H at ER/ PM contact sites [41] and TMEM16K at the ER/ endosome interface [43]. Also in plant cells, the ER forms copious connections with other organelles [44], and the subcellular distribution of lipids, membrane signalling at the nano level and interorganellar communication are active fields of research [45,46]. With the identification of a probable lipid scrambling activity for AtTMEM16, a new player has entered the field. Pollen tube protoplasts from tmem16 knockout plants showed altered anion currents [26], a phenotype possibly due to indirect effects. In the light of the findings presented here, it will be interesting to screen the knockout plants for specific defects related to protein trafficking, lipid homeostasis and signalling.

cDNA constructs
The full-length AtTMEM16 coding sequence was amplified from Arabidopsis thaliana Col-0 cDNA. Total RNA was isolated from leaves of 12-week-old Col-0 plants (SV Total RNA Isolation System, Promega, Milano, Italy) and retrotranscribed into cDNA. The PCR-amplification product (oligonucleotides P1-fw and P1-rv; Table S1) was subcloned into the pGEM-T Easy vector (Promega). Sequence analysis confirmed its identity with the At1g73020 gene coding sequence (GenBank NM_105960) deposited at TAIR (The Arabidopsis Information Resource).
For transient expression in Arabidopsis protoplasts, translational fusion constructs with C-terminal EGFP and N-terminal YFP were prepared. For the EGFP fusion, the AtTMEM16 coding sequence was PCR amplified without its stop codon using the oligonucleotides P1-fw and P2-rv. This modified AtTMEM16 ORF was inserted into the unique EcoRI cloning site of the plant expression vector pSAT6-EGFP-N1 [48]. For the YFP fusion, the AtTMEM16coding sequence was PCR amplified using the oligonucleotides P2-fw and P1-rv. The EcoRI-digested PCR product was inserted into the unique EcoRI cloning site of the plant expression vector pSAT3228-YFP (kindly provided by Alex Costa, University of Milan, Italy), which puts the AtTMEM16 ORF in frame with YFP. The correct orientation of the insert was verified by restriction digest and ORF continuity was confirmed by sequencing. For transient expression in HEK293 cells, the AtTMEM16-EGFP cassette in the pSAT6-derived expression plasmid was PCR amplified using the oligonucleotides P3-fw and P3-rv and inserted into NotI/XbaI-digested pFROG expression vector [49].
The chimeric TMEM16A cDNA carrying the SCRD of AtTMEM16 (amino acids 366-400) was constructed using an overlap extension PCR approach. The stretch encoding AtTMEM16 SCRD was amplified using the oligonucleotides T16A_atSCRD_fw and T16A_atSCRD_rv (PCR I). The TMEM16A regions upstream and downstream of the SCRD were amplified using the oligonucleotide pairs T16A_5AclI-fw/atSCRD_T16A-rv (PCR II) and atSCRD_T16A-fw/T16A_3BspEI-rv (PCR III) respectively. The AtTMEM16 SCRD was linked to the TMEM16A upstream region using the products of PCR I and II as template and the oligonucleotide pairs T16A_5AclI_fw/ T16A_atSCRD_rv (PCR IV). The TMEM16A downstream region was then added using the products of PCR III and IV as template and the oligonucleotide pairs T16A_5AclI_fw/T16A_3BspEI-rv. Finally, the resulting chimeric DNA fragment was inserted into the AclI/BspEI restriction sites of the pEGFP-TMEM16A plasmid, replacing the native TMEM16A fragment.
Single amino acid exchanges were generated using specifically designed oligonucleotides and the QuickChange Lightning site-directed mutagenesis kit (Agilent Technologies, Cernusco Sul Naviglio, Italy). Mutagenesis was confirmed by DNA sequencing.

Protoplast isolation and transient transformation
Plants of Arabidopsis thaliana (ecotype Columbia-0) were grown on soil in a growth chamber at 22°C and under an 8 h light/ 16 h dark regime. Mesophyll protoplasts were isolated from well-expanded rosette leaves of 4-to 8-weekold plants. Leaf tissue was digested in enzyme solution containing 1% cellulase and 0.2% macerozyme for 3 h at 23°C. The protoplast suspension was filtered through a 50lm nylon mesh, washed and used for polyethylene glycol (PEG) transformation at a density of 2 9 10 5 cells ml À1 [51,52]. Protoplasts transformed with either pSAT6-AtTMEM16-EGFP or pSAT3228-YFP-AtTMEM16 For a quantitative assessment of AtTMEM16 colocalization with fluorescently labelled ER marker proteins, we determined Manders' M coefficient representing the fraction of AtTMEM16-positive pixels containing ERmarker-derived signal [54]. M values range from 0 (no overlap) to 1 (complete overlap). Image analysis was done using the JACoP plugin [55] in IMAGEJ 1.53 K (at https:// imagej.nih.gov/ij/) [56].
In HEK293 cells, endoplasmic reticulum was stained using CellLight ER-RFP BacMam 2.0 (Thermo Fisher Scientific, Monza, Italy), applied to the Petri dish 36 h before visualization. The PM marker FM4-64 (Thermo Fisher Scientific) was added to HEK293 cells or protoplasts at a final concentration of 10 lM, and cells were imaged immediately.
For lipid scrambling assays, HEK293 cells were washed in a buffer solution (140 mM NaCl, 2.5 or 5 mM CaCl 2 , 10 mM HEPES, pH 7.4) and incubated with Alexa fluor-555conjugated annexin-V (Thermo Fisher Scientific), at a dilution of 1 : 100-200, in the absence or presence of the Ca 2+ionophore A23187 (5-10 lM) for 5 min. Ionophore solution was prepared freshly from a 1-mM stock solution (in DMSO) stored at À20°C. Alexa fluor-555 was excited with the 543-nm laser line and emission acquired at 560-675 nm.

Patch-clamp electrophysiology
Current recordings on transiently transfected HEK293 cells were performed in the whole-cell patch-clamp configuration, as described elsewhere [15,30]. The extracellular solution contained (in mM): 140 NaCl, 5 K-gluconate, 2 CaSO 4 , 2 MgSO 4 , 10 HEPES, pH 7.4. 10 to 30 mM glucose was added to reduce volume-regulated chloride currents. The intracellular solution with 3 µM free Ca 2+ contained (in mM): 130 CsCl, 3.209 mM CaCl 2 , 10 HEPES, 10 HEDTA, pH 7.2. Free Ca 2+ was calculated using the program WinMAXC [57]. Stimulation protocols consisted of voltage steps of 300 ms duration ranging from À80 to +160 mV (with 20-mV increments), followed by a 175-ms tail pulse to À80 mV, from a holding potential of 0 mV. Current amplitudes were evaluated at the end of the test pulse, between 280 and 300 ms. Data analysis and figure preparation was done using Ana (freely available at http:// users.ge.ibf.cnr.it/pusch/programs-mik.htm) and Igor PRO SOFTWARE (Wavemetrics, Lake Oswego, OR, USA). For the sake of clarity, capacitative transients of current traces were trimmed in the figures.

Statistical analyses
Data are reported as mean values AE standard error of the mean. Statistical significance was determined using ANOVA followed by a post hoc Tukey's test to evaluate which data groups showed significant differences. P values < 0.05 were considered significant. analysis and interpretation of data, and in the writing of the report or in the decision to submit the article for publication. Open Access Funding provided by Consiglio Nazionale delle Ricerche within the CRUI-CARE Agreement. WOA Institution: Consiglio Nazionale delle Ricerche. Blended DEAL: CARE. Open Access Funding provided by Consiglio Nazionale delle Ricerche. [Correction added on 26 May 2022, after first online publication CRUI funding statement has been added.]

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. Sequence alignment of Arabidopsis and mammalian TMEM16 proteins. Table S1. Oligonucleotides used in this study.